Rapid Identification Of Pathogenic Bacteria By Means Of Two Conservable Gene Locuses Specific PCR-CE-RFLP

The rRNA genetic locus, rrn, is found in all prokaryotic organisms, and is highly conservable, although its relatively stable variation is found frequently in different bacteria. The utility of this locus as a taxonomic and phylogenetic tools had been reported widely. 16S rRNA and 16S-23S rRNA spacer region gene are the commonly used targets of this locus. When it comes to 16S rRNA, many scholars chose different primers to amplify certain parts of the gene by means of Polymerase Chain Reaction(PCR). In order to improve the efficacies of identification, they utilized Single-Strand Conformation Polymorphism(SSCP) technology. Although most of the studied bacteria could be distinguished by different peak patterns correctly, there were still some ones belonging to the most common genera, e.g., E.coli and Shigella ssp. could not be separated from each other. Then another gene -16S-23S rRNA spacer region gene, was introduced to facilitate the identification of microorganisms. After the argarose gel electrophoresis of PCR product of this gene, the patterns of different bacteria showed distinct sizes and/or numbers polymorphism. But this method was too much time consuming and it could not give a definite numerized size lengths of the fragments. The application to clinical use was very limited. For the genus of staphylococcus, its results were too complex to explain. In order to utilize these two genes efficiently, we choose two pairs of primers aiming at 16S rRNA and 16S-23S rRNA spacer region appropriately, and label them with two kinds of fluorescence -FAM and JOE, respectively. We also simplify the template preparation procedures and modify the PCR reaction condiction with shorter annealing time and extention time, which allow the two reactions can take place simultaneously. The PCR product was then digested by restriction endonuclease Hae III incompletely for 30 minutes. The mixture solutions were diluted for 50 times before take capillary electrophoresis, and the Restriction-Fragment-Length Polymorphism(RFLP) were detected by ABI Prism 310Genetic Analyzer. This study is aiming to construct an RFLP database of common pathogenic bacteria in case that it be used in future to facilitate the microorganisms' identification from clinical samples. We collected 183 gram's positive and negative strains belonging to 5 genera and 19 species, whose metabolism characterization are in accord on the behaviors of the typical ones. After through analysis of PCR-CE-RFLP of 16S rRNA gene, the data could only be used to classify the bacteria into some families. According to the information come from the 16S-23S rRNA spacer region gene, we could identify the whole organisms as definitely as the spices level. All the bacteria that could not be distinguished from each other by the first two methods mentioned above could be identified correctly. In spite of the data of the spacer region gene alone can be sufficiently to verify the whole microorganisms, we insist that the 16S rRNA gene be of great assistance in the condition that when we are confronted with the big families of microorganism, in which some similar ones ,with the same RFLP data of 16S-23s rRNA spacer region gene ,may coexist. This study prove that the use of PCR-CE-RFLP of 16S rRNA and 16S-23S rRNA spacer region genes is a convenient, rapid method to identify pathogenic bacteria, and is also a quick diagnosis measure of great application for clinical use.